Cell Penetrating Peptide Conjugates for Delivering of Nucleic Acids into a Cell

Abstract

The invention provides cell penetrating peptide-nucleic acid conjugates having the formula P-L-N, wherein P is a cell penetrating peptide, N is a nucleic acid, preferably an oligonucleotide and more preferably a siRNA, and L is a hydrophilic polymer, preferably a polyethylene glycol (PEG)-based linker linking P and N together. Compositions, methods of use and methods for producing such conjugates are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is the U.S. National Stage filing of International Application No. PCT / IB2006 / 003642, filed Dec. 15, 2006, which claims priority to EP 05292722.5, filed Dec. 16, 2005 and U.S. Provisional Patent Application No. 60 / 755,053, filed Jan. 3, 2006, the disclosures of each of which are hereby incorporated by reference.BACKGROUND OF THE INVENTION[0002](i) Field of the Invention[0003]The invention relates generally to delivery of nucleic acids using cell penetrating peptides. More particularly it concerns cell penetrating peptide-nucleic acid conjugates enabling an efficient delivery of said nucleic acid into cells both in vitro and in vivo.[0004](ii) Description of the Related Art[0005]Genetic information now available from the human genome sequence may be exploited for the design of specific agents to modulate the function of genes or their protein products to correct genetic disorders or to treat diseases, such as cancers. A nu...

AI Technical Summary

Benefits of technology

[0021]This invention specifically has as its object to offer new cell penetrating peptide-nucleic acid conjugates with improved delivery efficacy into cells both in vitro and in vivo.

[0234]Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

Problems solved by technology

However, these strategies are hampered by the relatively inefficient delivery of genetic material into either somatic or cultured cells.

These methods all have limitations generally with regard to: efficiency of delivery, low concentration of nucleic acid delivered into the target cell or organ to give a biological effect in vivo, potential size-limit of the nucleic acid being transported, safety concerns and / or production scale-up difficulties.

The major limitation of siRNA application, as for antisense RNA or nucleic acid-based strategies, remains their poor cellular uptake related to low permeability of the cell membrane to nucleic acids (Luo and Saltzman, Nat. Biotechnol. 18:33-37 (2000); Niidome and Huang, Gene Ther.

However, in many cases, when one tries to conjugate basic amino acid-rich peptides to an oligonucleotide in solution-phase, the reaction can not be carried out because of serious precipitation caused by electrostatic interaction of the two moieties.

Also the formation of such heterogenous products is not compatible with therapeutic development.

However, applying this procedure for the synthesis of conjugates containing an amino acid sequence longer than 10 amino acids or an oligonucleotide sequence longer than 10 nucleotides could be difficult to due to the low yield of such a procedure.

Further, this procedure doesn't allow a combinatorial approach for screening multiple oligonucleotide-PEG-peptide conjugates in parallel.

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